Ammonia-oxidizing bacteria (AOB) are very sensitive to environmental conditions and wastewater treatment plant operational parameters. One of the most important factors affecting their activity is pH. Its effect is associated with: NH3/NH4+ and HNO2/NO2− chemical equilibriums and biological reaction rates. The aim of this study was to quantify and model the effect of pH and free nitrous acid (FNA) concentration on the activity of AOB present in a lab-scale partial nitritation reactor. For this purpose, two sets of batch experiments were carried out using biomass from this reactor. Fluorescent in situ hybridization analysis showed that Nitrosomona eutropha and Nitrosomona europaea species were dominant in the partial nitritation reactor (>94%). The experimental results showed that FNA inhibits the AOB activity. This inhibition was properly modelled by the non-competitive inhibition function and the half inhibition constant value was determined as 1.32 mg HNO2-N L−1. The optimal pH for these AOB was found to be in the range 7.4–7.8. The pH inhibitory effect was stronger at high pH values than at low pH values. Therefore, an asymmetric inhibition function was proposed to represent the pH effect on these bacteria. A combination of two sigmoidal functions was able to reproduce the experimental results obtained.